US8334522B2 - Method for the quantitative determination of the concentration of fluorophores of a substance in a sample and apparatus for carrying out the same - Google Patents
Method for the quantitative determination of the concentration of fluorophores of a substance in a sample and apparatus for carrying out the same Download PDFInfo
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- US8334522B2 US8334522B2 US12/616,811 US61681109A US8334522B2 US 8334522 B2 US8334522 B2 US 8334522B2 US 61681109 A US61681109 A US 61681109A US 8334522 B2 US8334522 B2 US 8334522B2
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- sample
- reference light
- receiving element
- optical element
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
- G01N21/278—Constitution of standards
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Definitions
- the invention concerns a method for the quantitative determination of the concentration of fluorophores of at least one substance in a sample, wherein this substance is irradiated with light of an excitation wave length emitted by an excitation light source and the intensity of the fluorescent light of an emission wave length coming from the sample is measured by means of a receiving element.
- fluorescence standard for calibrating a measured intensity value of the fluorescent light, which standard emits fluorescent light of a known wave length distribution and intensity when irradiated with excitation light of a preset wave length and intensity.
- a method and an apparatus for the determination of the fluorescence of a test sample are for instance known from the document EP 0 237 363 A2.
- an optical element which couples in a constant portion of a reference light of a reference wave length emitted by a reference light source in the direction of a receiving element. This portion is constant for the optical element also over longer periods of time so that this optical element is suited to constitute a standard of comparison for evaluating a measured value of the detected fluorescent light.
- a first measured value is detected which corresponds to the portion of the reference light coupled in by the optical element which is incident on the receiving element.
- the sample is irradiated with excitation light of an excitation wave length emitted by an excitation light source.
- a second measured value is detected which corresponds to the portion of the fluorescent light of an emission wave length emitted by the sample.
- the relationship of the second measured value and the first measured value is determined. The number of fluorophores within a detection zone present in the substance of the sample is determined taking this relationship into account.
- the measured value obtained during the detection of the fluorescent light is thus put into relation to the measured value of the reference light coupled in by the optical element so that the optical element serves as a reference object or reference standard.
- a single basic adjustment of the optical element serving as the standard of comparison to a fluorescence standard with a known number of fluorophores within a detection zone can be carried out.
- a corrected measured value or the number of fluorophores, respectively can then simply be determined by relating to the optical element.
- a measuring apparatus for determining the number of fluorophores in the substance of a sample within the detection zone.
- the number of fluorophores in the substance of the sample is determined within the detection zone of the sample with the aid of the detected relationship and with the aid of the relationship of a third measured value and of a fourth measured value detected during a calibration of the measuring apparatus.
- the constant portion of the reference light emitted by the reference light source is coupled in by the optical element in the direction of the receiving element.
- a third measured value is detected which corresponds to the portion of the light coupled in which is incident on the receiving element.
- a fluorescence standard is irradiated with the excitation light emitted by the excitation light source.
- a fourth measured value is detected which corresponds to the portion of the fluorescent light emitted by the fluorescence standard which is incident on the receiving element.
- the relationship between the fluorescence standard and the optical element is determined during the calibration process in a simple manner so that the detected measured values of a sample can be related to the optical element. Based on the relationship between the optical element and the fluorescence standard detected during the calibration it is then possible to determine the number of fluorophores of the sample or a corrected measured value which corresponds to the fluorescent light incident on the receiving element.
- the optical element is preferably a reflectance standard. Due to its optical properties, such a reflectance standard reflects a constant portion of the incident light at least in one wave length range. Due to this, the reflectance standard can be positioned in lieu of a sample or in lieu of the fluorescence standard and/or besides a sample or besides a fluorescence standard for detecting the measured values.
- the number of fluorophores of the sample is preferably determined using the following formula:
- scaling factor X can be determined using the following equation:
- the intensity of the reference light emitted by the reference light source is also advantageous to measure with the help of a further receiving element. Also the intensity of the excitation light emitted by the excitation light source can be measured with the help of this further receiving element. The relationship between the intensity of the excitation light and the intensity of the reference light is then taken into account when determining the number of fluorophores of the sample. Due to this, it is not necessary to use light sources with constant respective light emission, but the light emission of the light sources can vary within a certain range since changes in the light emission are taken into account when evaluating the detected fluorescent light emitted by the sample.
- optical element as a standard of comparison for the calibration of the detected second measured value, whereby in particular contamination, temperature effects and/or deterioration effects of elements of a measuring apparatus for carrying out the method can be compensated with the help of the standard of comparison.
- the optical path between the reference light source and the optical element used for coupling in the constant portion of the reference light emitted by the reference light source in the direction of the receiving element to pass through the same optical elements as the optical path of the excitation light between the excitation light source and the sample or between the excitation light source and the fluorescence standard, respectively.
- an arrangement for carrying out the method can have a simple construction.
- changes in the properties of the optical elements in the optical path affect both the reference light and the excitation light.
- an additional filter may be arranged whose transmission range is centered about the excitation wave length. This filter is not positioned within the optical path of the reference light between the reference light source and the optical element.
- the optical path between the optical element serving for coupling in the constant portion of the reference light emitted by the reference light source in the direction of the receiving element and the receiving element to pass through the same optical elements as the optical path of the fluorescent light between the sample and the receiving element or between the fluorescence standard and the receiving element, respectively.
- changes in the optical properties of the optical elements affect the reference light and the fluorescent light in the same way.
- the factor X can be used preferably in measuring arrangements comprising a stabilized reference light source and a stabilized excitation light source which each emit reference light or excitation light, respectively, with a constant intensity.
- the sample and the optical element can be preferably measured by means of a scanning method. Alternately also a non-scanning measuring of the sample and the optical element is possible.
- An apparatus for carrying out the method preferably includes a carrier for a sample to be measured, an emission branch comprising an excitation light source for emitting excitation light of an excitation wave length, a first receiving element for measuring the intensity of the excitation light and a first filter arranged within the path of the excitation light, the transmission range of which filter is centered about the excitation wave length, as well as a receiving branch comprising a second receiving element for measuring the intensity of the fluorescent light of an emission wave length coming from the sample, and a second filter whose transmission range is centered about the emission wave length.
- a reference light source for emitting at least one reference beam of the emission wave length is arranged within the emission branch.
- the first receiving element serves to measure the intensity of the light of both light sources of the emission branch.
- an optical element is arranged so that it receives the light of the reference light source of the emission branch.
- filters are used for this purpose, which are chosen such that a filter arranged within the excitation light beam generally only transmits the wave length of the excitation light while a filter arranged in the measuring branch generally only transmits the wave length of the fluorescent light. For this reason in an advantageous embodiment of the solution according to the invention reference light of the emission wave length, i.e. the wave length of the fluorescent light, is used for measuring the optical element.
- the two light sources of the emission branch can be arranged so that the optical paths of the light beams of the two light sources of the emission branch are at least approximately identical. However, they may instead be arranged in such a way that the optical paths of the excitation light and of the reference light are directed onto the reflectance standard under different angles.
- the reference light source has to have a higher intensity, since the reference light also passes through the filter arranged within the excitation light beam whose transmission range corresponds in general to the excitation wave length and thus considerably attenuates the intensity of the reference light of the emission wave length. This is avoided in the second case.
- a calibration can be carried out with the help of an optical element or of a reflectance standard which can be produced with the desired long-term stability. It is sufficient to carry out a single adjustment of this optical element with the help of an adjusted fluorescence standard during a starting-up calibration of a measuring apparatus for carrying out the method. Subsequently the fluorescence density of a sample can always be determined with reference to a deposited internal optical element or reflectance standard.
- the starting-up calibration is for example carried out at the place of manufacture of the measuring apparatus before delivering the measuring apparatus to the customer or alternately during a start-up or reconnection of the measuring apparatus at the location of the customer or user.
- the aim of such a starting-up calibration is in particular to minimize a variation of the measuring results between several produced measuring apparatus and/or to enable traceability of the measuring results of a measuring apparatus to an authoritative standard with long-term stability or to a collective of several measuring apparatus.
- the measuring results of all measuring apparatus of the collective can be compared to each other in a simple manner.
- the apparatus can be modified to include further features which are indicated in particular with respect to the method according to the invention.
- the apparatus can be modified to include the features of the dependent method claims or corresponding apparatus features.
- FIG. 1 shows the wave length distribution of the excitation light and of the fluorescent light as well as the filter characteristics of the respective interference filters for the excitation light and the fluorescent light
- FIGS. 2 and 3 show schematic depictions of two embodiments of measuring arrangements for carrying out the method according to the invention.
- FIGS. 4 and 5 show depictions of two further embodiments of the emission branch of an apparatus for carrying out the method according to the invention.
- FIG. 1 shows the gradient of efficiency of the excitation light with reference to the wave length by means of a graph 10 , as well as the emission characteristic of the corresponding fluorescent light by means of a graph 12 .
- a rectangle 14 represents the filter characteristic of the excitation branch or emission branch
- the rectangle 16 represents the filter characteristic of a receiving or measuring branch.
- interference filter with very steep edges are preferably used as filters 14 and 16 .
- the interference filters for the excitation branch are preferably dimensioned such that maximum excitation efficiency is reached and the wave length of the excitation light source is within the transmission range of the filter.
- reference number 18 denotes schematically a carrier for a sample to be measured or a reflectance standard, respectively.
- an optical module generally indicated at 20 , is arranged which comprises an emission branch 22 and a receiving or measuring branch 24 .
- the emission branch 22 comprises a first light source or excitation light source 26 , which emits light of the excitation wave length ⁇ ex .
- a second light source or reference light source 28 which emits light of the reference wave length ⁇ em .
- This wave length generally corresponds to the wave length of the fluorescent light emitted by the excited sample and thus lies within the filter characteristic 16 .
- the two light sources can be LEDs.
- the beams of both light sources 26 and 28 pass through a filter 30 which has the filter characteristic 14 of FIG. 1 . Since the reference wave length ⁇ em lies outside the filter characteristic 14 , the reference light is attenuated by a factor of 100 to 1,000,000.
- the output power of the reference light source is preferably considerably higher than that of the excitation light source 26 .
- the intensities of both light sources 26 and 28 are read by a monitor diode presenting a first receiving element 32 .
- the receiving branch 24 has an optics 34 , a filter 36 with the filter characteristic 16 , as well as a photo diode 38 serving as a second receiving element.
- a reflectance standard is measured.
- the reference beam emitted by the reference light source 28 is used for this purpose.
- the wave length ⁇ em of the reference light is attenuated by the filter 30 .
- the reflectance of the reflectance standard reaches the photo diode 38 without obstructions. Since the reflectance is higher by several orders of magnitude than the fluorescence, a major part of the attenuation of the reference light by the filter 30 is compensated for.
- the performance at the photo diode 38 can therefore be compared to the performance of the fluorescence emission of the sample.
- the monitor diode 32 however is activated considerably less by the reference light source 28 than by the excitation light source 26 .
- measuring methods In an integrated measuring method this can be partially compensated for by means of different lengths of integration times. Also measuring methods exist which provide a measuring range of about 200 even with constant integration times at a signal-to-noise ratio of 100. A combination of different integration times and measuring ranges should make it possible to reach an intensity difference of the two light sources at the monitor diode 32 of about 50,000 with a signal-to-noise ratio of 100.
- the actual fluorescence measurement is performed with light of the wave length ⁇ ex .
- the calibration of the measuring results is carried out by referencing them to the measuring of the internal standard.
- the beam paths of the reference light and of the excitation light should be nearly identical as is the case in the embodiment of FIG. 1 .
- the light paths within the receiving branch are nearly identical since during the excitation of the fluorescence the emitted wave length corresponds to the reference wave length.
- the respective projection is identical.
- the transmission by the filter 36 can vary slightly, since the spectrum of the emission is broader than the spectrum of the reference light source. However, this is a constant which does not change with time. Also the temperature gradation is insignificantly small.
- the light of the reference light source 28 is fed into a light conductor 40 separately from the excitation light source 26 .
- the light conductor 40 homogenizes the light so that the respective intensity distributions of the two light sources are almost identical at the outlet.
- a portion of the light is coupled out by means of a beam splitter 42 and directed to the monitor diode 32 .
- the beam splitter 42 can also be omitted and the light can be coupled out separately from the light conductor 40 and directed to the monitor diode 32 .
- the light distribution is nearly identical for the two wave lengths ⁇ ex and ⁇ em at the point 44 on the sample or on the reflectance standard, respectively.
- the advantage of the embodiment according to FIG. 3 as compared to the embodiment according to FIG. 2 lies in the fact that the reference light does not pass through the filter 30 and is consequently not attenuated. Therefore a weaker reference light source may be used.
- the relationship of the light intensities at the monitor diode 32 and at the photo diode 38 can be controlled via the output power of the reference light source.
- FIG. 4 shows only the emission branch 22 of the measuring arrangement.
- the light beams of both light sources 26 and 28 are being collimated by respective lenses 46 .
- the light beams are only combined with each other upon reaching the measuring plane 18 .
- the filter 30 is only arranged in the excitation light beam.
- the reference light reaches the measuring plane 18 without being attenuated by a filter.
- the performance of both light sources is again measured by means of a monitor diode 32 which is arranged directly in the illumination beam path. However, this does not impede the illumination of the measuring field whose size is determined via an aperture 48 .
- the projection optics, i.e. the receiving branch 24 is not shown in this case.
- the optical axis of the receiving branch is tilted in the x-z-plane, while the optical axis of illumination is tilted in the z-y-plane as shown in FIG. 4 .
- FIG. 5 shows an embodiment which is preferably used in non-scanning systems.
- the measuring field is completely illuminated and the fluorescent light is imaged onto a two-dimensional sensor.
- the receiving branch is not shown.
- a diffusion disk 50 is used. Otherwise the construction of this embodiment is similar to the embodiment according to FIG. 4 . However, here the diffusion disk 50 and not the measuring field is illuminated directly. Due to this construction, the illumination distribution in the measuring plane 18 should be almost identical for both wave lengths ⁇ ex and ⁇ em .
- the internal standard is measured with the help of the reference light source before the actual measurement takes place.
- the remission measurement can be carried out with a QC-element (quality cartridge) which instead of the fluorescence measuring field has a defined remission.
- the QC-measurement is then not carried out before each measurement, with this measure deterioration effects of the light source or contamination of the optics can be identified early on.
- E mes1 P ex1 ⁇ FD FS ⁇ K ex
- the factor K ex indicates how efficiently the performance of the excitation wave length ⁇ ex is transformed into fluorescence. Since normally not the entire performance of the illumination in the measuring field plane is measured by the monitor diode 32 , also the ratio of measured performance to total performance is included in this factor.
- a condition for further calculation is that the relationship between K em and K ex remains constant. Since only the relationship between P ex and P em is relevant for the following constant X and not their absolute values, this assumption should be correct. Contamination of the optics e.g. affects K ex and K em in the same way so that the constant X is not influenced by it.
- the measurements P em2 and E s2 are determined.
- the values E mes2 and P ex2 are measured.
- the constant X′ and the value REM of the reflectance standard are known.
- the receiving elements mentioned above may be photo diodes, line sensors, area sensors or also photo-multipliers. In scanning systems preferably photodiodes and line sensors are used.
- the photodiode 32 can also be used for stabilizing the performance of the light sources.
- the optical output performance normally is reduced due to heating processes during operation. These effects can be reduced by performing a corresponding loop control. Also deterioration processes of the LEDs can be compensated for in this manner.
- the test strips or sample carriers are thus always irradiated with the same power and possible bleaching always occurs under the same conditions.
- laser diodes as a rule comprise an internal monitor diode, which can be used for performance control, the stability which can be reached in this manner is often not sufficient.
Abstract
Description
wherein
- FDP denotes the number of fluorophores of the sample within the detection zone;
- Emes2 denotes a second measured value corresponding to the portion of the fluorescent light of an emission wave length coming from the sample and incident on the receiving element;
- Es2 denotes a first measured value corresponding to the portion of the light coupled in which is incident on the receiving element; and
- X denotes a constant scaling factor which represents a relationship between the optical element used and the fluorescence standard and which is determined for the optical element during the calibration of the measuring apparatus.
wherein
- Es1 denotes a third measured valued corresponding to the constant portion of the reference light emitted by the reference light source and coupled in by the optical element in the direction of the receiving element during the calibration and incident on the receiving element;
- Emes1 denotes a fourth measured value corresponding to the portion of the fluorescent light emitted by the fluorescence standard which is incident on the receiving element;
- FDFS denotes the known number of fluorophores of the fluorescence standard within the detection zone.
E mes1 =P ex1 ·FD FS ·K ex
-
- wherein
- Emes1 denotes the measured intensity of the fluorescent light emitted by the fluorescence standard;
- Pex1 denotes the measured intensity of the excitation light;
- FDFS denotes the number of the fluorophores of the fluorescence standard within the detection zone; and
- Kex denotes a proportionality constant;
to subsequently measure the optical element by means of the reference light using the following equation:
E s1 =P em1 ·REM·K em - wherein
- Es1 denotes the measured intensity of the reference light coupled in by the optical element in the direction of the receiving element;
- Pem1 denotes the measured intensity of the reference light emitted by the reference light source;
- REM denotes the constant portion of the reference light coupled in by the optical element in the direction of the receiving element; and
- Kem denotes a proportionality constant;
and to calculate X′ from the above using the following equation:
X′ differs from X in that the measured light intensities of the excitation light and of the reference light are taken into account as well. Thus the factor X can be used preferably in measuring arrangements comprising a stabilized reference light source and a stabilized excitation light source which each emit reference light or excitation light, respectively, with a constant intensity.
- 1. The fluorescence standard FDFS is used as a comparison standard for the starting-up calibration during the start-up of each single measuring apparatus. This requires that this fluorescence standard is highly stable or that it can be verified with the help of a reference device and that the changes in the properties of the fluorescence standard detected during verification are being taken into account during the starting-up calibration.
- 2. The fluorescence standard FDFS is used when starting up a reference measuring apparatus. Then an optical element, in particular a reflectance standard, is measured with the help of the reference measuring apparatus and a relationship between the fluorescence standard and the optical properties of the optical element, in particular the reflectance properties of the reflectance standard, is determined. In this way, a definite correlation between the reflectance values of the optical element and the fluorescence properties of the fluorescence standard FDFS is determined which is then preset in further measuring apparatus as the correlation between the reflectance values of optical elements provided in these further measuring apparatus. The optical elements of the further measuring apparatus have the same optical reflectance properties as the optical element of the reference measuring apparatus. For determining the fluorescence properties of a sample to be examined, the scaling factor X or X′ is then used as has been explained in detail above. The fluorescence standard FDFS is not necessarily an indicator of the number of fluorophores per surface area or the fluorophore density. Instead, FDP can be a general measure of the concentration of a substance within a sample.
E mes1 =P ex1 ·FD FS ·K ex
wherein
-
- Pex1 =the intensity of the excitation light measured with the help of the
monitor diode 32; - FDFS=the number of fluorophores of the fluorescence standard within the detection zone;
- Emes1=the intensity of the fluorescent light emitted by the fluorescence standard and measured with the help of the
photo diode 38; and - Kex=a proportionality constant to be determined using the above equation.
- Pex1 =the intensity of the excitation light measured with the help of the
E s1 =P em1 ·REM·K em
wherein
-
- Pem1=the intensity of the reference light measured with the help of the
monitor diode 32; - REM=the constant portion of the reference light coupled in by the optical element in the direction of the receiving
element 38; - Es1=the measured intensity of the reference light coupled in by the optical element in the direction of the receiving
element 38; - Kem=a proportionality constant to be determined using the above equation.
- Pem1=the intensity of the reference light measured with the help of the
K ex =X·K em
Claims (18)
E mes1 =P ex1 ·FD FS ·K ex
E s1 =P em1 ·REM·K em
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DE102008057115A DE102008057115B4 (en) | 2008-11-13 | 2008-11-13 | Method for the quantitative determination of the concentration of fluorophores of a substance in a sample and apparatus for carrying it out |
DE102008057115 | 2008-11-13 | ||
DE102008057115.6 | 2008-11-13 |
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US20100117003A1 US20100117003A1 (en) | 2010-05-13 |
US8334522B2 true US8334522B2 (en) | 2012-12-18 |
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US10197545B2 (en) | 2015-07-29 | 2019-02-05 | Advanced Sensors Limited | Method and apparatus for measurement of a material in a liquid through absorption of light |
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DE102011002080B4 (en) * | 2011-04-15 | 2016-05-04 | Lre Medical Gmbh | Apparatus and method for determining the concentration of fluorophores in a sample |
DE102011111315A1 (en) * | 2011-08-26 | 2013-02-28 | Bundesrepublik Deutschland, vertreten durch das Bundesministerium für Wirtschaft und Technologie, dieses vertreten durch den Präsidenten der Physikalisch-Technischen Bundesanstalt | Method for fluorescence measurement |
JP6280770B2 (en) * | 2013-03-27 | 2018-02-14 | オリンパス株式会社 | measuring device |
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Also Published As
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US20100117003A1 (en) | 2010-05-13 |
DE102008057115B4 (en) | 2013-11-28 |
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